The enteric nervous system (ENS) is the division of the autonomic nervous system that resides within the gut wall. The ENS controls gastrointestinal (GI) motility, secretion and local blood flow. The ENS can perform these complex functions because it contains all the neuronal elements (sensory neurons, interneurons and motorneurons) required for GI reflexes and integration. The ENS contains 14 different types of neurons that release different neurotransmitters. There are also multiple receptors for each neurotransmitter. In addition, synapses in the ENS may be coded by the neurotransmitters released from presynaptic nerve terminals and by receptors expressed by postsynaptic cells. The proposed studies will use intracellular electrophysiological, immunohistochemical and molecular biological methods to study enteric neuromuscular transmission. There are 3 specific aims in this proposal. Specific aim 1 will test the hypothesis that there are two separate populations of inhibitory nerves supplying the muscle layers. One subset uses nitric oxide (NO) as the primary neurotransmitter while the second population is purinergic (ATP and/or b-nicotinamide adenine dinucleotide are the neurotransmitters). These studies will show that release of ATP/b-NAD and NO from nerve terminals is controlled by different Ca2+ channel types. An antibody against the vesicular nucleotide (VNUT) antibody will be used to localize purinergic nerves. These studies will also make use of P/Q type and R-type Ca2+ channel mutant mice. Specific aim 2 will focus on Ca2+ channels expressed by interneurons in the myenteric plexus. Interneurons which project in an oral-anal direction release acetylcholine (ACh) and ATP as fast synaptic transmitters, while neurons that project in an anal-oral direction release ACh. These studies will test the hypothesis that R-, N- and P/Q type Ca2+ channels are expressed by neurons in the orally-projecting pathway while only N- and P/Q type Ca2+ channels are expressed by nerve terminals in the anally-projecting pathway. These studies will also use wild type and P/Q-type and R-type Ca2+ channel mutant mice. Specific aim 3 will focus on K+ channels as regulators of gut smooth muscle tone and neuromuscular transmission in the colon. These studies will make use of a b1 subunit of the large conductance Ca2+-activated K+ (BK) channel knockout mouse. Significance: Disturbances in enteric synaptic mechanisms contribute to GI motility disorders. Changes in the function of enteric neurons and their synapses might also contribute to visceral pain. Therefore, a more complete understanding of enteric neural circuits and synaptic transmission would provide insights into the pathophysiology of GI motility disorders. This information would help to develop new drug treatments for common motility disorders.